www.sciencesignaling.org/cgi/content/full/7/313/ra17/DC1

Supplementary Materials for

The Adaptor Protein p66Shc Inhibits mTOR-Dependent Anabolic Metabolism

Mohamed A. Soliman,* Anas M. Abdel Rahman, Dudley A. Lamming, Kivanç Birsoy,

Judy Pawling, Maria E. Frigolet, Huogen Lu, I. George Fantus, Adrian Pasculescu, Yong Zheng, David M. Sabatini, James W. Dennis,* Tony Pawson

*Corresponding author. E-mail: ma.soliman@utoronto.ca (M.A.S.); dennis@lunenfeld.ca (J.W.D.)

Published 18 February 2014, Sci. Signal. 7, ra17 (2014)

DOI: 10.1126/scisignal.2004785 This PDF file includes:

Fig. S1. A volcano plot for the metabolomic analysis of HeLa cells stably expressing shRNA targeting GFP or p66Shc. Fig. S2. Unsupervised principal components analysis for targeted metabolomic analysis of p66Shc KO and p66+ MEFs. Fig. S3. The abundance of GLUT1 in p66Shc-competent and p66Shc-deficient HeLa cells and MEFs is similar. Fig. S4. p66Shc inhibits de novo synthesis of nonessential amino acids. Fig. S5. Effect of amino acid deprivation on serum-mediated activation of mTORC1 in p66Shc-deficient cells. Fig. S6. Effect of p66Shc expression on cell size. Fig. S7. Effect of Akt inhibition on the amounts of glycolytic metabolites in p66Shc-competent and p66Shc-deficient MEFs. Legend for table S1 Table S2. LC-MS/MS transitions for [1,2-13C2]glucose intermediates. Table S3. List of genes showing differential regulation upon p66Shc expression. Table S4. List of identified p66Shc-interacting proteins.

Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/7/313/ra17/DC1)

Table S1. LC-MS/MS transitions for the metabolites measured in this study (Excel file).

Fig. S1. A volcano plot for the metabolomics analysis of HeLa cells stably expressing

shRNA targeting GFP or p66Shc. Significance is represented on the y-axis (-log10(p)) and fold

change of p66Shc-competent over p66Shc-deficient HeLa cells is represented on the x-axis

(log2). Representative metabolites with significant changes are labelled. N = 3 independent

biological replicates.

Fig. S2. Unsupervised principal component analysis for targeted metabolomic analysis of

p66Shc KO and p66+ MEFs. Each dot represents a biological replicate. Red dots indicate

replicates for p66Shc KO MEFs, and green dots indicate replicates for p66+ MEFs. The analysis

demonstrates clear separation of overall metabolomic profiles of the two genotypes with

statistical significance 99.7% (PC1) and 0.2% (PC2) (n=3, p < 0.05). The metabolites with the

most significant changes in p66+ MEFs compared to KO MEFs are summarized in Table 1.

A

B

Fig. S3. The abundance of GLUT1 in p66Shc-competent and p66Shc-deficient HeLa cells

and MEFs is similar. (A) HeLa cells stably transfected with indicated shRNAs were lysed and,

and cell lysates were analyzed for the indicated proteins by immunoblotting. N = 2 independent

biological replicates. (B) Lysates of p66Shc KO and p66+ MEFs were examined as in (A). N = 2

independent biological replicates.

Fig. S4. p66Shc inhibits de novo synthesis of nonessential amino acids. Fold change of 13C-

labeled serine (A) and alanine (B) in p66Shc KO (white) and p66+ (black) MEFs. Error bars

represent SD of at least three independently prepared samples (*** p < 0.001).

M2 Alanine

KO p66+0.0

0.5

1.0

1.5

Fo

ld c

han

ge

M2 Serine

KO p66 +0.0

0.5

1.0

1.5

Fo

ld c

han

ge

A B

*** ***

Fig. S5. Effect of amino acid deprivation on serum-mediated activation of mTORC1 in

p66Shc-deficient cells. (A) HeLa cells stably transfected with shRNAs targeting the indicated

genes were starved of amino acids for 50 min and stimulated either with dialyzed serum or

normal serum for 10 min. Phosphorylation of S6K1 was analyzed by Western blotting. N = 3

independent biological replicates. (B) p66Shc KO and p66+ MEFs were treated and analyzed as

in (A). N = 3 independent biological replicates.

Fig. S6. Effect of p66Shc expression on cell size. (A) Cell size measurement of KO and p66+

cells. Cell size was measured using coulter counter. N = 3 biological replicates. (B) Cell size

measurement of p66Shc–deficient and p66Shc-competent HeLa cells. Experiment was done as in

(A). N = 3 biological replicates.

A

B

Fig. S7. Effect of Akt inhibition on the amounts of glycolytic metabolites in p66Shc-

competent and p66Shc-deficient MEFs. (A-D) Fold change of 3PG (A), PEP (B), R5P (C),

and citrate (D) in p66Shc KO MEFs (white), p66Shc KO MEFs treated with Akt inhibitor

(black), p66+ (grey) and p66

+ treated with Akt inhibitor (stripped) MEFs. Error bars represent

SD of at least three independently prepared samples (p < 0.05, *** p < 0.001).

***

***

A B

C D

***

***

***

***

***

*

Table S1. LC-MS/MS transitions for the metabolites measured in this study (Excel file). DP: de-clustering potential, CE: Collision energy, HMDB: Human Metabolome Database.

Table S2. LC-MS/MS Transitions for [1,2-13C2]glucose intermediates. DP: de-clustering

potential, EP: entrance potential, CE: Collision energy, CXP: collision cell exit potential

Q1 Q3 RT ID DP CE CXP

87 43 7.41

Pyruvate -35 -14 -3

89 45 [1,2]13C-Pyruvate [M2] -35 -14 -3

89 43

7.41

Lactate -55 -16 -1

90 44 [1]13C-lactate [M1] -55 -16 -1

91 45 [1,2]13C-lactate [M2] -55 -16 -1

191 111 8.6

Citrate -30 -18 -7

193 112 [1,2]13C-Citrate [M2] -30 -18 -7

259 97 7.33 G6P 97 -40 -28 -5

261 79 7.33

[1,2]13C-G6P [M2] -40 -28 -5

261 97 [1,2]13C-G6P [M2] -40 -28 -5

259 79 8.26 D-Fructose-6P -55 -72 -11

261 79 8.26 [1,2]13C-Fructose-6P [M2] -55 -72 -11

300 79 7.7

GlcNAcP -45 -76 -15

302 79 [1,2]13C-GlcNAcP [M2] -60 -76 -15

606 159 8.5

UDP-GlcNAc -110 -66 -1

608 159 UDP-[1,2]13C-GlcNAc [M2] -110 -66 -1

808 159

8.5

Acetyl Co-enzyme A (ACoA) -150 -88 -9

809 159 [2]13C-Acetyl Co-enzyme A (ACoA) [M1]

-150 -88 -9

810 159 [1,2]13C-Acetyl Co-enzyme A (ACoA) [M2]

-150 -88 -9

Table S3: List of genes showing differential regulation upon p66Shc expression.

Gene Gene full Name p66Shc KO - p66+(average log

10)

p-value SD

Lrp2 low density lipoprotein receptor-related protein 2

-1.915792811 0.00053 0.265341548

Lingo4 leucine rich repeat and Ig domain containing 4

-1.811543741 0.00053 0.08564836

Fam19a2 family with sequence similarity 19, member A2

-1.635935145 0.00053 0.094915819

Fras1 Fraser syndrome 1 homolog (human)

-1.632833069 0.00053 0.088349326

Epha7 Eph receptor A7 -1.617034208 0.00053 0.265811188

2810405K02Rik RIKEN cDNA 2810405K02 gene -1.545497146 0.00053 0.104072241

Pcdhgb8 protocadherin gamma subfamily B, 8

-1.505347374 0.00053 0.134934987

Nol4 nucleolar protein 4 -1.491293872 0.00053 0.147634015

Elavl2 ELAV (embryonic lethal, abnormal vision, Drosophila)-like 2 (Hu antigen B)

-1.475961437 0.00053 0.172756757

Slc38a4 solute carrier family 38, member 4 -1.450549921 0.00053 0.082508643

Slc16a7 solute carrier family 16 (monocarboxylic acid transporters), member 7

-1.422033949 0.00053 0.180917562

Fam132b family with sequence similarity 132, member B

-1.408791619 0.00053 0.067851145

Fndc3c1 fibronectin type III domain containing 3C1

-1.317312614 0.00053 0.082532254

Cpa6 carboxypeptidase A6 -1.311506175 0.00053 0.39594187

Lrrtm1 leucine rich repeat transmembrane neuronal 1

-1.30232534 0.00053 0.261829654

Tmem178 transmembrane protein 178 -1.229814213 0.00053 0.252731011

Pcdh9 protocadherin 9 -1.209521591 0.00053 0.06012591

Rnf128 ring finger protein 128 -1.200148584 0.00053 0.295306529

Mfap3l microfibrillar-associated protein 3-like

-1.184275636 0.00053 0.471271669

Nr3c2 nuclear receptor subfamily 3, group C, member 2

-1.179205418 0.00053 0.245215328

Epha3 Eph receptor A3 -1.141945788 0.00053 0.18094104

Pde3b phosphodiesterase 3B, cGMP-inhibited

-1.126724114 0.00053 0.058435767

Armcx4 armadillo repeat containing, X-linked 4

-1.118182406 0.00053 0.009426341

Pcsk5 proprotein convertase subtilisin/kexin type 5

-1.110683672 0.00053 0.122133709

Mycn v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)

-1.109619722 0.00053 0.085221891

Fibin fin bud initiation factor homolog (zebrafish)

-1.075052764 0.00053 0.053954081

Mei4 meiosis-specific, MEI4 homolog (S. cerevisiae)

-1.027144733 0.00054 0.404083607

Slc26a7 solute carrier family 26, member 7 -1.018616084 0.00053 0.265390359

Pmaip1 phorbol-12-myristate-13-acetate-induced protein 1

-0.991734978 0.00053 0.180923943

Sfrp2 secreted frizzled-related protein 2 -0.974846673 0.00053 0.172783959

Tll1 tolloid-like -0.957443259 0.00053 0.175013394

Tmem151b transmembrane protein 151B -0.952357544 0.00053 0.148567314

Tnfrsf21 tumor necrosis factor receptor superfamily, member 21

-0.92950432 0.00053 0.067073319

Lef1 lymphoid enhancer binding factor 1 -0.929191993 0.00054 0.311455724

Hunk hormonally upregulated Neu-associated kinase

-0.925378588 0.00054 0.488512276

Tdrkh tudor and KH domain containing protein

-0.924298141 0.00053 0.016466993

Plcl1 phospholipase C-like 1 -0.922570232 0.00053 0.050416995

Pkia protein kinase inhibitor, alpha -0.917178399 0.00053 0.145255127

Tm6sf2 transmembrane 6 superfamily member 2

-0.909526811 0.00054 0.265433652

Mtap7d3 MAP7 domain containing 3 -0.905721395 0.00054 0.265381819

Zfp583 zinc finger protein 583 -0.89964319 0.00054 0.231461609

Fam38b family with sequence similarity 38, member B

-0.886354776 0.00054 0.393893458

Sort1 sortilin 1 -0.883128964 0.00053 0.036959691

Hist1h2bg histone cluster 1, H2bg -0.870775698 0.00054 0.08807413

Snca synuclein, alpha -0.850845519 0.00054 0.101000827

Dok5 docking protein 5 -0.829479412 0.00054 0.098944847

Rell2 RELT-like 2 -0.824736054 0.00054 0.261803243

Gabre gamma-aminobutyric acid (GABA) A receptor, subunit epsilon

-0.824104256 0.00054 0.134844879

Hist1h4k histone cluster 1, H4k -0.821945916 0.00054 0.296458294

Gm10406 predicted gene 10406 -0.808209064 0.00054 0.180904117

Cacna1b calcium channel, voltage-dependent, N type, alpha 1B

-0.805835164 0.00054 0.143616773

subunit

Hoxb9 homeobox B9 -0.797828438 0.00054 0.198343907

Asxl3 additional sex combs like 3 (Drosophila)

-0.791955705 0.00054 0.054836257

Pcdhb2 protocadherin beta 2 -0.787980234 0.00054 0.160613469

Snx10 sorting nexin 10 -0.784827431 0.00054 0.100231686

Pcdhb3 protocadherin beta 3 -0.779873929 0.00054 0.114090528

4921528I01Rik RIKEN cDNA 4921528I01 gene -0.777509771 0.00054 0.192939047

Wnt2b wingless related MMTV integration site 2b

-0.755864987 0.00054 0.0903634

Peg10 paternally expressed 10 -0.744421658 0.00054 0.10331376

Fndc5 fibronectin type III domain containing 5

-0.744227916 0.00054 0.074817703

Ntng2 netrin G2 -0.741595235 0.00054 0.121176342

Klf12 Kruppel-like factor 12 -0.738830144 0.00054 0.041258524

Npy1r neuropeptide Y receptor Y1 -0.736048621 0.00054 0.125603467

Gpr137c G protein-coupled receptor 137C -0.73215916 0.00059 0.534608914

8430408G22Rik RIKEN cDNA 8430408G22 gene -0.724087278 0.00055 0.222465861

Bhlhe22 basic helix-loop-helix family, member e22

-0.718249387 0.00054 0.150545533

Nlrx1 NLR family member X1 -0.713254896 0.00054 0.030703712

Elovl4 elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 4

-0.711290071 0.00055 0.223211263

Chdh choline dehydrogenase -0.705962742 0.00056 0.361115226

Sema6a sema domain, transmembrane domain (TM), and cytoplasmic

-0.703651997 0.00054 0.059363453

domain, (semaphorin) 6A

Cthrc1 collagen triple helix repeat containing 1

-0.701838551 0.00055 0.173849535

Enox1 ecto-NOX disulfide-thiol exchanger 1

-0.699117306 0.00054 0.114088181

Fbxl7 F-box and leucine-rich repeat protein 7

-0.698448854 0.00054 0.044721972

Snrpn small nuclear ribonucleoprotein N -0.696287165 0.00055 0.123683742

1700010I14Rik RIKEN cDNA 1700010I14 gene -0.687747012 0.00056 0.2527405

Tmem169 transmembrane protein 169 -0.686456758 0.00057 0.395917183

Palm3 paralemmin 3 -0.684145211 0.00059 0.50659648

Tnfaip8l1 tumor necrosis factor, alpha-induced protein 8-like 1

-0.683349349 0.00055 0.029609797

Cd200 CD200 antigen -0.681445884 0.00055 0.046093045

Pcdh8 protocadherin 8 -0.680995051 0.00056 0.27531672

Dclk2 doublecortin-like kinase 2 -0.676081914 0.00055 0.134788259

Igf2 insulin-like growth factor 2 -0.674359709 0.00055 0.036664029

Fut4 fucosyltransferase 4 -0.672636529 0.00057 0.404091035

F2rl1 coagulation factor II (thrombin) receptor-like 1

-0.664956186 0.00055 0.016152716

Tmem200a transmembrane protein 200A -0.663179322 0.00055 0.036468114

Tbx2 T-box 2 0.663640332 0.00055 0.021998753

Gm1661 predicted gene 1661 0.665248537 0.00055 0.108950542

Diras2 DIRAS family, GTP-binding RAS-like 2

0.667441057 0.00056 0.246125103

Rab3b RAB3B, member RAS oncogene family

0.667673045 0.00055 0.087214478

Col28a1 collagen, type XXVIII, alpha 1 0.668464877 0.00055 0.103303578

Agt angiotensinogen (serpin peptidase inhibitor, clade A, member 8)

0.668787912 0.00055 0.059258563

Slc22a23 solute carrier family 22, member 23

0.670209459 0.00055 0.036892269

Il13ra2 interleukin 13 receptor, alpha 2 0.670898039 0.00055 0.077902592

Syt5 synaptotagmin V 0.673105204 0.00055 0.134876587

Cfh complement component factor h 0.673403902 0.00055 0.014587192

Cdh26 cadherin-like 26 0.674748099 0.00055 0.171407963

1700034H15Rik RIKEN cDNA 1700034H15 gene 0.676046005 0.00056 0.339792831

Lgals9 lectin, galactose binding, soluble 9 0.678298366 0.00055 0.051080452

Gm14393 predicted gene 14393 0.680908099 0.00058 0.403850786

Adh7 alcohol dehydrogenase 7 (class IV), mu or sigma polypeptide

0.68327306 0.00055 0.152894719

Cygb cytoglobin 0.683903613 0.00055 0.157736829

4-Sep septin 4 0.684369152 0.00061 0.376028174

Nsg1 neuron specific gene family member 1

0.68443605 0.00055 0.027139644

Tmem117 transmembrane protein 117 0.6868859 0.00055 0.123397066

Siglecg sialic acid binding Ig-like lectin G 0.687365882 0.00055 0.072380418

Cbln3 cerebellin 3 precursor protein 0.688270313 0.00055 0.066008871

Islr immunoglobulin superfamily containing leucine-rich repeat

0.692047166 0.00054 0.019871733

Adamtsl1 ADAMTS-like 1 0.695033204 0.00054 0.104145348

Lama3 laminin, alpha 3 0.69513384 0.00054 0.108472544

Acy3 aspartoacylase (aminoacylase) 3 0.696801941 0.00054 0.086061403

Atf3 activating transcription factor 3 0.69773903 0.00054 0.024900306

Rsph1 radial spoke head 1 homolog (Chlamydomonas)

0.701830671 0.00055 0.207766755

Phactr1 phosphatase and actin regulator 1 0.703918435 0.00054 0.055588964

9930023K05Rik RIKEN cDNA 9930023K05 gene 0.704413698 0.00058 0.462960081

Atp8b1 ATPase, class I, type 8B, member 1

0.704855288 0.00054 0.066155661

Timp3 tissue inhibitor of metalloproteinase 3

0.705980282 0.00054 0.009519119

Ticam2 toll-like receptor adaptor molecule 2

0.706980197 0.00055 0.216094637

Nhsl2 NHS-like 2 0.708152301 0.00055 0.187820581

Tcf7 transcription factor 7, T cell specific

0.710676527 0.00054 0.079743011

Asgr1 asialoglycoprotein receptor 1 0.710908571 0.00054 0.102195858

Gem GTP binding protein (gene overexpressed in skeletal muscle)

0.711129665 0.00054 0.074775012

Acss1 acyl-CoA synthetase short-chain family member 1

0.713136814 0.00056 0.332812454

Adrbk2 adrenergic receptor kinase, beta 2 0.71389249 0.00054 0.017370486

Mapkapk3 mitogen-activated protein kinase-activated protein kinase 3

0.717198672 0.00054 0.11169346

Map3k5 mitogen-activated protein kinase kinase kinase 5

0.722403796 0.00054 0.060276991

Islr2 immunoglobulin superfamily containing leucine-rich repeat 2

0.723526789 0.00054 0.137727559

Ggt7 gamma-glutamyltransferase 7 0.724810409 0.00054 0.121828837

Zfp296 zinc finger protein 296 0.726254956 0.00059 0.440252288

Sh2d1b1 SH2 domain protein 1B1 0.726373223 0.00054 0.086700286

B3gnt8 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 8

0.729573708 0.00057 0.303349205

Tnni3 troponin I, cardiac 3 0.731254977 0.00058 0.514112037

9130019O22Rik RIKEN cDNA 9130019O22 gene 0.732389495 0.00054 0.098879186

Ccbe1 collagen and calcium binding EGF domains 1

0.733942913 0.00054 0.137946031

Gstt1 glutathione S-transferase, theta 1 0.734574564 0.00054 0.095911655

Rtp4 receptor transporter protein 4 0.737249757 0.00054 0.050153752

Abi3 ABI gene family, member 3 0.739334553 0.00054 0.101934592

Aldh1a3 aldehyde dehydrogenase family 1, subfamily A3

0.742734742 0.00054 0.085524474

Rasl11a RAS-like, family 11, member A 0.743417171 0.00054 0.069964518

Epb4.1l4a erythrocyte protein band 4.1-like 4a

0.743720766 0.00054 0.067778459

Ica1 islet cell autoantigen 1 0.746449159 0.00058 0.532783978

Hyal3 hyaluronoglucosaminidase 3 0.746670131 0.00058 0.532544673

Ifi27l2a interferon, alpha-inducible protein 27 like 2A

0.748805838 0.00054 0.128921448

Matn4 matrilin 4 0.751296432 0.00055 0.309099514

Afap1l2 actin filament associated protein 1-like 2

0.754643517 0.00054 0.040200803

Abcc3 ATP-binding cassette, sub-family C (CFTR/MRP), member 3

0.755485412 0.00055 0.246203752

Deptor DEP domain containing MTOR-interacting protein

0.758137134 0.00054 0.116223933

Smoc1 SPARC related modular calcium binding 1

0.759456057 0.00056 0.432661364

Dcn decorin 0.759916709 0.00054 0.063105273

Cldn1 claudin 1 0.761833728 0.00054 0.04408684

Traf1 TNF receptor-associated factor 1 0.764048952 0.00054 0.065079109

Cfb complement factor B 0.76455062 0.00054 0.088308557

Vtcn1 V-set domain containing T cell activation inhibitor 1

0.765821423 0.00055 0.267925708

Acta1 actin, alpha 1, skeletal muscle 0.765964795 0.00054 0.043835532

Gm12216 predicted gene 12216 0.76644448 0.00054 0.135900469

1700003F12Rik RIKEN cDNA 1700003F12 gene 0.768055495 0.00054 0.089105998

Saa3 serum amyloid A 3 0.773580491 0.00054 0.173053452

Cdhr1 cadherin-related family member 1 0.774388432 0.00054 0.045177575

Ptgfr prostaglandin F receptor 0.775513945 0.00054 0.090611927

Susd2 sushi domain containing 2 0.775594375 0.00054 0.097255044

Cdkn1c cyclin-dependent kinase inhibitor 1C (P57)

0.776172936 0.00054 0.06379327

Mndal myeloid nuclear differentiation antigen like

0.779296909 0.00055 0.379651909

Clec11a C-type lectin domain family 11, member a

0.780058561 0.00054 0.055581965

Apol10b apolipoprotein L 10B 0.782834626 0.00054 0.137041661

Cyp26b1 cytochrome P450, family 26, subfamily b, polypeptide 1

0.782836703 0.00054 0.117528419

Kctd14 potassium channel tetramerisation domain containing 14

0.784766049 0.00056 0.360430947

AI428936 expressed sequence AI428936 0.787294696 0.00054 0.099279262

Adam33 a disintegrin and metallopeptidase domain 33

0.788839092 0.00054 0.085605242

Aox1 aldehyde oxidase 1 0.790952518 0.00054 0.028503273

Blnk B cell linker 0.790963356 0.00054 0.301575058

Ecscr endothelial cell surface expressed chemotaxis and apoptosis regulator

0.793488682 0.00054 0.035874271

Rarres2 retinoic acid receptor responder (tazarotene induced) 2

0.793997302 0.00054 0.137749917

Epas1 endothelial PAS domain protein 1 0.794441813 0.00054 0.093825829

Fam179a family with sequence similarity 179, member A

0.797152443 0.00054 0.085500996

Sncg synuclein, gamma 0.797621076 0.00054 0.089113939

Angptl7 angiopoietin-like 7 0.79815192 0.00054 0.044478057

Eln elastin 0.798869667 0.00054 0.018790951

Gprin3 GPRIN family member 3 0.800688631 0.00054 0.128589472

Rhox5 reproductive homeobox 5 0.801127842 0.00055 0.265045427

Pcdhga2 protocadherin gamma subfamily A, 2

0.802342483 0.00055 0.4339809

Ppl periplakin 0.806596571 0.00054 0.017650785

Pdzrn4 PDZ domain containing RING finger 4

0.816216272 0.00054 0.03802449

Bdh1 3-hydroxybutyrate dehydrogenase, type 1

0.818013017 0.00054 0.13315328

Gbp6 guanylate binding protein 6 0.820104623 0.00054 0.059587685

Auts2 autism susceptibility candidate 2 0.823419711 0.00054 0.017035484

Peg3 paternally expressed 3 0.824601124 0.00054 0.095374777

Slfn2 schlafen 2 0.828174735 0.00054 0.180890243

Espnl espin-like 0.829729488 0.00054 0.187549219

Raet1d retinoic acid early transcript delta 0.831300419 0.00054 0.084477615

Col10a1 collagen, type X, alpha 1 0.834622555 0.00054 0.043812899

Usp18 ubiquitin specific peptidase 18 0.841243699 0.00054 0.33200192

Rab40b Rab40b, member RAS oncogene family

0.845386805 0.00054 0.216744132

Ddit4l DNA-damage-inducible transcript 4-like

0.846972376 0.00054 0.038527775

Panx1 pannexin 1 0.847590486 0.00054 0.159315477

Isg15 ISG15 ubiquitin-like modifier 0.856119102 0.00054 0.114701267

Lxn latexin 0.85697897 0.00054 0.065921894

Cercam cerebral endothelial cell adhesion molecule

0.860725077 0.00054 0.094089887

Ankrd6 ankyrin repeat domain 6 0.861965839 0.00054 0.148669382

Naalad2 N-acetylated alpha-linked acidic dipeptidase 2

0.863873157 0.00054 0.043166595

Ceacam1 carcinoembryonic antigen-related cell adhesion molecule 1

0.868657678 0.00054 0.09876767

Nptx1 neuronal pentraxin 1 0.868733414 0.00055 0.432899662

Tnfrsf9 tumor necrosis factor receptor superfamily, member 9

0.869749964 0.00054 0.0822082

Apol6 apolipoprotein L 6 0.870822486 0.00054 0.096801623

Thsd4 thrombospondin, type I, domain containing 4

0.874631254 0.00054 0.375134475

Pcdhb13 protocadherin beta 13 0.877924773 0.00054 0.085463636

Acsbg1 acyl-CoA synthetase bubblegum family member 1

0.878723314 0.00054 0.30885885

Serpina3n serine (or cysteine) peptidase inhibitor, clade A, member 3N

0.879110484 0.00053 0.041690783

Aoc3 amine oxidase, copper containing 3 0.881136113 0.00053 0.018828905

5430407P10Rik RIKEN cDNA 5430407P10 gene 0.881624059 0.00053 0.072724759

Serpina3m serine (or cysteine) peptidase inhibitor, clade A, member 3M

0.883754442 0.00053 0.060574541

Bst1 bone marrow stromal cell antigen 1 0.886785141 0.00056 0.540574601

Stac2 SH3 and cysteine rich domain 2 0.892804709 0.00054 0.147549381

Gjb3 gap junction protein, beta 3 0.893450437 0.00055 0.431657445

Nap1l3 nucleosome assembly protein 1-like 3

0.893947626 0.00054 0.139544026

Kng2 kininogen 2 0.895607628 0.00053 0.074813929

Il33 interleukin 33 0.896838083 0.00054 0.144757383

Ngfr nerve growth factor receptor (TNFR superfamily, member 16)

0.898674402 0.00053 0.085524089

Sorl1 sortilin-related receptor, LDLR class A repeats-containing

0.899089977 0.00054 0.152335302

H2-T10 histocompatibility 2, T region locus 10

0.902292843 0.00054 0.380676837

Insc inscuteable homolog (Drosophila) 0.90417236 0.00053 0.112156662

Spink2 serine peptidase inhibitor, Kazal type 2

0.905591442 0.00054 0.14676946

Clstn3 calsyntenin 3 0.908201748 0.00053 0.089411025

Gpr4 G protein-coupled receptor 4 0.909466117 0.00054 0.216203667

Cxcl17 chemokine (C-X-C motif) ligand 17

0.909828619 0.00055 0.537563025

Abcg1 ATP-binding cassette, sub-family G (WHITE), member 1

0.91071001 0.00054 0.274435175

Plcd4 phospholipase C, delta 4 0.913182402 0.00054 0.171352782

Ifit1 interferon-induced protein with tetratricopeptide repeats 1

0.918782586 0.00053 0.175317001

Cd74 CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated)

0.926534365 0.00053 0.095906798

Rhpn2 rhophilin, Rho GTPase binding protein 2

0.927138782 0.00053 0.08551548

Dgkg diacylglycerol kinase, gamma 0.927520067 0.00053 0.100662246

Adh1 alcohol dehydrogenase 1 (class I) 0.928183835 0.00054 0.359441834

Edar ectodysplasin-A receptor 0.928254958 0.00053 0.072900312

Il2rb interleukin 2 receptor, beta chain 0.928805896 0.00054 0.267954993

Aim1 absent in melanoma 1 0.929351782 0.00053 0.057430612

Prex2 phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2

0.93719502 0.00053 0.08997535

Ptk2b PTK2 protein tyrosine kinase 2 beta

0.938135831 0.00053 0.027228824

Ldhd lactate dehydrogenase D 0.93871423 0.00054 0.439647844

1700024P16Rik RIKEN cDNA 1700024P16 gene 0.938819604 0.00054 0.360716271

Gstt3 glutathione S-transferase, theta 3 0.948569948 0.00053 0.024634112

Sim2 single-minded homolog 2 (Drosophila)

0.953991468 0.00053 0.143385491

Efhd1 EF hand domain containing 1 0.956252223 0.00053 0.103256292

Oasl2 2'-5' oligoadenylate synthetase-like 2

0.959954864 0.00053 0.023324485

Neurl3 neuralized homolog 3 homolog (Drosophila)

0.964136118 0.00053 0.134849684

Kng1 kininogen 1 0.977131516 0.00053 0.049991385

Sp7 Sp7 transcription factor 7 0.978273933 0.00054 0.309076084

Parm1 prostate androgen-regulated mucin-like protein 1

0.980508708 0.00053 0.058662293

Foxf2 forkhead box F2 0.981038713 0.00053 0.059632285

C1qtnf5 C1q and tumor necrosis factor related protein 5

0.982903387 0.00054 0.290011907

Spib Spi-B transcription factor (Spi-1/PU.1 related)

0.983279881 0.00053 0.157542051

Fgd4 FYVE, RhoGEF and PH domain containing 4

0.984260836 0.00053 0.139318882

D14Ertd668e DNA segment, Chr 14, ERATO Doi 668, expressed

0.988864035 0.00053 0.127634024

Dll1 delta-like 1 (Drosophila) 0.992361465 0.00054 0.432629062

Rnase1 ribonuclease, RNase A family, 1 (pancreatic)

0.993085648 0.00054 0.285009166

Acox2 acyl-Coenzyme A oxidase 2, branched chain

0.993184757 0.00053 0.094325878

Cybrd1 cytochrome b reductase 1 0.994028526 0.00053 0.225943878

Sult1a1 sulfotransferase family 1A, phenol-preferring, member 1

0.995373222 0.00053 0.12120022

Tfap2a transcription factor AP-2, alpha 0.997539572 0.00053 0.135302675

Pdgfb platelet derived growth factor, B polypeptide

0.998420792 0.00053 0.163272181

Hck hemopoietic cell kinase 0.999070587 0.00053 0.250495191

C2cd4a C2 calcium-dependent domain containing 4A

0.999639773 0.00053 0.208161313

Pcdhgb1 protocadherin gamma subfamily B, 1.005347499 0.00053 0.080214417

1

9030224M15Rik RIKEN cDNA 9030224M15 gene 1.013271323 0.00054 0.359742465

Mustn1 musculoskeletal, embryonic nuclear protein 1

1.015777183 0.00053 0.127381615

Tbxa2r thromboxane A2 receptor 1.015799552 0.00053 0.044372957

Rapsn receptor-associated protein of the synapse

1.017472248 0.00053 0.106670772

Mgst1 microsomal glutathione S-transferase 1

1.018475319 0.00053 0.0203985

Gas6 growth arrest specific 6 1.018597236 0.00053 0.012972127

Ptprq protein tyrosine phosphatase, receptor type, Q

1.023762028 0.00053 0.09260246

Mrvi1 MRV integration site 1 1.024551664 0.00053 0.047280673

Selenbp1 selenium binding protein 1 1.027124543 0.00053 0.136774659

H28 histocompatibility 28 1.027624929 0.00053 0.114131969

Pcdhga6 protocadherin gamma subfamily A, 6

1.027721531 0.00053 0.273393221

Serpina3f serine (or cysteine) peptidase inhibitor, clade A, member 3F

1.031536232 0.00053 0.178581046

Ccl27a chemokine (C-C motif) ligand 27A 1.03329066 0.00054 0.484304318

Iigp1 interferon inducible GTPase 1 1.036102758 0.00053 0.065017448

Apol9b apolipoprotein L 9b 1.039598534 0.00053 0.153952589

Mmp13 matrix metallopeptidase 13 1.040888988 0.00053 0.083124011

Fmo4 flavin containing monooxygenase 4 1.046495119 0.00053 0.216815067

Steap4 STEAP family member 4 1.052428065 0.00053 0.002584317

Cln3 ceroid lipofuscinosis, neuronal 3, juvenile (Batten, Spielmeyer-Vogt

1.058161466 0.00053 0.309026893

disease)

C1qtnf3 C1q and tumor necrosis factor related protein 3

1.062870795 0.00053 0.085885934

Dio2 deiodinase, iodothyronine, type II 1.07779171 0.00053 0.209791746

Tmprss6 transmembrane serine protease 6 1.079375769 0.00053 0.033600766

Xdh xanthine dehydrogenase 1.085912708 0.00053 0.079133761

Pcdhgb6 protocadherin gamma subfamily B, 6

1.088637837 0.00053 0.059579985

Alox5ap arachidonate 5-lipoxygenase activating protein

1.09044588 0.00053 0.166524406

Notum notum pectinacetylesterase homolog (Drosophila)

1.10405039 0.00053 0.094076015

Lbp lipopolysaccharide binding protein 1.113722934 0.00053 0.03739157

Clec3b C-type lectin domain family 3, member b

1.116350535 0.00053 0.076252528

Enpp2 ectonucleotide pyrophosphatase/phosphodiesterase 2

1.117753237 0.00053 0.347134136

Acpp acid phosphatase, prostate 1.120588722 0.00053 0.137820192

Rragb Ras-related GTP binding B 1.125262389 0.00053 0.309078515

Gdnf glial cell line derived neurotrophic factor

1.132545993 0.00053 0.109641032

Hp haptoglobin 1.13791102 0.00053 0.43130165

Lcn2 lipocalin 2 1.139064261 0.00053 0.08005039

Hspa12b heat shock protein 12B 1.14116058 0.00053 0.085648791

Syt13 synaptotagmin XIII 1.143486055 0.00053 0.027679077

Slc16a2 solute carrier family 16 (monocarboxylic acid transporters),

1.159110478 0.00053 0.398843774

member 2

Sod3 superoxide dismutase 3, extracellular

1.167418756 0.00053 0.056023357

Ugt1a6b UDP glucuronosyltransferase 1 family, polypeptide A6B

1.170555987 0.00053 0.128208789

Slc22a18 solute carrier family 22 (organic cation transporter), member 18

1.174411334 0.00053 0.145295089

Entpd3 ectonucleoside triphosphate diphosphohydrolase 3

1.180120689 0.00053 0.1037217

Cxcl10 chemokine (C-X-C motif) ligand 10

1.193671012 0.00053 0.140252908

Zfp941 zinc finger protein 941 1.194086034 0.00053 0.085421735

Elfn1 leucine rich repeat and fibronectin type III, extracellular 1

1.194848088 0.00053 0.020379081

Apol9a apolipoprotein L 9a 1.197174371 0.00053 0.246164347

Gpbar1 G protein-coupled bile acid receptor 1

1.198285099 0.00053 0.135408239

Cda cytidine deaminase 1.205054123 0.00053 0.237088505

Fam107a family with sequence similarity 107, member A

1.205425335 0.00053 0.039519945

Nr1h3 nuclear receptor subfamily 1, group H, member 3

1.206406134 0.00053 0.091558614

Plcg2 phospholipase C, gamma 2 1.206711745 0.00053 0.398864245

Trim30a tripartite motif-containing 30A 1.208785913 0.00053 0.144143522

Csmd1 CUB and Sushi multiple domains 1 1.210273167 0.00053 0.057302114

Psca prostate stem cell antigen 1.212806007 0.00053 0.091731027

Psmb9 proteasome (prosome, macropain) subunit, beta type 9 (large

1.213436685 0.00053 0.1345473

multifunctional peptidase 2)

Pcsk6 proprotein convertase subtilisin/kexin type 6

1.216391075 0.00053 0.032624856

Prelp proline arginine-rich end leucine-rich repeat

1.216786271 0.00053 0.051151785

A4galt alpha 1,4-galactosyltransferase 1.22310685 0.00053 0.061975306

Vnn1 vanin 1 1.22704685 0.00053 0.016872923

Fxyd1 FXYD domain-containing ion transport regulator 1

1.231112772 0.00053 0.523075014

Trpv2 transient receptor potential cation channel, subfamily V, member 2

1.248179355 0.00053 0.216250134

Renbp renin binding protein 1.248781807 0.00053 0.017383204

Grhl1 grainyhead-like 1 (Drosophila) 1.253278296 0.00053 0.270603819

Msl3l2 male-specific lethal 3-like 2 (Drosophila)

1.25986456 0.00053 0.137146309

Arhgap6 Rho GTPase activating protein 6 1.266639625 0.00053 0.180941556

Ccl5 chemokine (C-C motif) ligand 5 1.300214635 0.00053 0.131616389

H60b histocompatibility 60b 1.301586423 0.00053 0.311046603

H2-Q4 histocompatibility 2, Q region locus 4

1.303535724 0.00053 0.084559364

C3 complement component 3 1.322856021 0.00053 0.063090714

AI607873 expressed sequence AI607873 1.346856052 0.00053 0.137647255

Cyp2j9 cytochrome P450, family 2, subfamily j, polypeptide 9

1.359477206 0.00053 0.086144262

Tnfsf10 tumor necrosis factor (ligand) superfamily, member 10

1.375656394 0.00053 0.185884401

C130074G19Rik RIKEN cDNA C130074G19 gene 1.377624185 0.00053 0.13747556

Serping1 serine (or cysteine) peptidase inhibitor, clade G, member 1

1.386331531 0.00053 0.095017679

Nfe2l3 nuclear factor, erythroid derived 2, like 3

1.389071601 0.00053 0.032589724

Gda guanine deaminase 1.400864825 0.00053 0.026537201

Crabp1 cellular retinoic acid binding protein I

1.404406104 0.00053 0.242432756

Htatip2 HIV-1 tat interactive protein 2, homolog (human)

1.407270603 0.00053 0.057078139

Plb1 phospholipase B1 1.44324694 0.00053 0.269786455

Oas2 2'-5' oligoadenylate synthetase 2 1.443352045 0.00053 0.137723246

Inmt indolethylamine N-methyltransferase

1.460243408 0.00053 0.310783802

Pik3r5 phosphoinositide-3-kinase, regulatory subunit 5, p101

1.467004844 0.00053 0.08535067

Pdzk1ip1 PDZK1 interacting protein 1 1.474467624 0.00053 0.042997429

Mmp3 matrix metallopeptidase 3 1.479116108 0.00053 0.058432453

Tril TLR4 interactor with leucine-rich repeats

1.50993966 0.00053 0.137914316

Tubg2 tubulin, gamma 2 1.527010062 0.00053 0.309441049

Pdpn podoplanin 1.555027038 0.00053 0.4399842

Sp140 Sp140 nuclear body protein 1.570560246 0.00053 0.250312212

Apod apolipoprotein D 1.61198472 0.00053 0.221408291

Chi3l1 chitinase 3-like 1 1.636684153 0.00053 0.583086161

Unc93b1 unc-93 homolog B1 (C. elegans) 1.66968256 0.00053 0.360200982

H2-DMb1 histocompatibility 2, class II, locus Mb1

1.67565171 0.00053 0.359347716

Slc15a2 solute carrier family 15 (H+/peptide transporter), member 2

1.700204294 0.00053 0.385075496

Wnt4 wingless-related MMTV integration site 4

1.713137287 0.00053 0.360869216

Fmo2 flavin containing monooxygenase 2 1.815536475 0.00053 0.308692687

Sparcl1 SPARC-like 1 1.816996676 0.00053 0.063385442

Cyp2f2 cytochrome P450, family 2, subfamily f, polypeptide 2

1.83370284 0.00053 0.078208358

Klhl29 kelch-like 29 (Drosophila) 1.88351542 0.00053 0.138028824

Eml2 echinoderm microtubule associated protein like 2

1.970482521 0.00053 0.027241317

Slpi secretory leukocyte peptidase inhibitor

2.08642332 0.00053 0.441915633

Lgals6 lectin, galactose binding, soluble 6 5.671892314 0.00053 0.198517766

Table S4: List of identified p66Shc-interacting proteins.

Identified protein No. of peptides

p66+ MEF – serum starvation

No. of peptides p66

+ MEF – serum

stimulation / 10 min

Shc1 900 920

Sgk269 22 23

FAM59A 4 27

Beta2-adaptin 5 23

Alpha1-adaptin 4 22

Grb2 0 12

Lrrk1 0 9

Anks1 1 7

PTPN12 1 5

Gab1 0 2

PP2C-beta 8 14

PP1A 2 2